IGF2BP2 promotes colorectal cancer progression by upregulating the expression of TFRC and enhancing iron metabolism

Abstract

Background: Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, ranking third for morbidity and mortality worldwide. At present, no effective control method is available for this cancer type. In tumor cells, especially iron metabolization, is necessary for its growth and proliferation. High levels of iron are an important feature to maintain tumor growth; however, the overall mechanism remains unclear.

Methods: We used western blotting, immunohistochemistry (IHC) and real-time quantitative PCR to analyze the expression of IGF2BP2 in cell lines and tissues. Further, RNA-sequencing, RNA immunoprecipitation and methylated RNA immunoprecipitation experiments explored the specific binding of target genes. Moreover, the RNA stability assay was performed to determine the half-life of genes downstream of IGF2BP2. In addition, the Cell Counting Kit-8, colony formation assay, 5-ethynyl-2'-deoxyuridine assay and flow cytometry were used to evaluate the effects of IGF2BP2 on proliferation and iron metabolism. Lastly, the role of IGF2BP2 in promoting CRC growth was demonstrated in animal models.

Results: We observed that IGF2BP2 is associated with iron homeostasis and that TFRC is a downstream target of IGF2BP2. Further, overexpression of TFRC can rescue the growth of IGF2BP2-knockdown CRC cells. Mechanistically, we determined that IGF2BP2 regulates TFRC methylation via METTL4, thereby regulating iron metabolism and promoting CRC growth. Furthermore, using animal models, we observed that IGF2BP2 promotes CRC growth.

Conclusion: IGF2BP2 regulates TFRC mRNA methylation via METTL4, thereby regulating iron metabolism and promoting CRC growth. Our study highlights the key roles of IGF2BP2 in CRC carcinogenesis and the iron transport pathways.

Keywords: Colorectal cancer; IGF2BP2; Iron metabolism; m6A-TFRC.

Fig. 1

IGF2BP2 is highly expressed in colorectal cancer and correlates with the tumor stage. (A, B) Differences in IGF2BP2 expression between COAD tissue and normal tissue were analyzed using the GEPIA database (http://gepia.cancer-pku.cn/detail.php). (C) IGF2BP2 expression in para-carcinoma tissues and COAD tumor tissues at various clinical stages as determined by IHC analysis. (D) IGF2BP2 expression in 50 COAD tumor tissue samples (10 samples were stage I, 24 were stage II, and 16 were stage III-IV). (Student’s t- test). (E) IGF2BP2 expression in colorectal cancer tissues and adjacent normal tissues (n = 50). (paired t- test). *P < 0.05 and **** P < 0.0001

Fig. 2

Downregulation of IGF2BP2 inhibits the proliferation and cell cycling of colorectal cancer cell lines. (A, B) HCT-116 and SW480 cells were transfected with IGF2BP2 siRNA, and then IGF2BP2 mRNA (A) and protein (B) levels were determined by qPCR and western blotting, respectively. (C-E) After IGF2BP2 silencing, the viability (C), colony forming ability (D), and proliferative ability (EdU assay) (E) of the cells were evaluated. Scale bars: 150 μm. (F, G, H) The effect of IGF2BP2 on the cell cycle. (F), HCT-116 and SW480 cells were transfected with the IGF2BP2 siRNA and subjected to flow cytometry after propidium iodide (PI) staining. After transfection, the cell cycle-related gene expression and protein levels were assessed using qRT-PCR (G) and western blotting (H), respectively. Each value represents the mean ± SD for triplicate samples (Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001

Fig. 3

IGF2BP2 promotes iron metabolism in colorectal cancer. (A) Gene Ontology (GO) analysis of the 20 relevant pathways in HCT-116 cells enriched using RNA-seq. (B) Intracellular iron levels were measured using an iron assay kit after transfecting HCT-116 and SW480 cell lines with IGF2BP2 siRNA. (C) Calcein-AM (C-AM) was used to quantify cellular LIP levels via spectrophotometric measurements at an absorbance wavelength of 525 nm. Subtract the mean fluorescence intensity (MFI) of C-AM from the MFI of C-AM treated with DFO. (D) ROS content in HCT-116 and SW480 cells were measured after IGF2BP2 knockdown. Each value represents the mean ± SD for triplicate samples (Student’s t-test). **P < 0.01, ***P < 0.001 and **** P < 0.0001

Fig. 4

IGF2BP2 participates in colorectal cancer growth by regulating the expression of iron metabolism-related genes. (A, B) qRT-PCR (A) and western blotting (B) of the potential target genes and their products in HCT-116 and SW480 cells after IGF2BP2 knockdown. (C) The decay rate of mRNA and qPCR of TFRC at the indicated time points after treating HCT-116 and SW480 cells with actinomycin D (5 µg/ml) after IGF2BP2 inhibition. (D) Differences in TFRC expression between COAD tissue and normal tissue were analyzed using the GEPIA ((http://gepia.cancer-pku.cn/detail.php)), UALCAN (http://ualcan.path.uab.edu/), TNMplot (https://tnmplot.com/analysis/) and UCSC (https://xena.ucsc.edu/) databases. (E) The expression of TFRC in para-carcinoma tissues and CRC tumor tissues at various clinical stages as determined via IHC analysis. (F) Expression of TFRC in different stages of colorectal cancer and adjacent normal tissues (n = 50). (G) TFRC expression in colorectal cancer tissues and adjacent normal tissues (n = 50). (H) Correlation between IGF2BP2 and TFRC expression in COAD in tissue microarrays. (I, J) Relative mRNA expression of METTL4, an iron metabolism gene, was determined using qRT-PCR (I) and western blotting (J). (K) m6A-RIP assay was performed to detect m6A levels in TFRC mRNA in HCT-116 cells after METTL4 silencing. (L) Binding of IGF2BP2 to TFRC was verified using the RIP assay. Each value represents the mean ± SD for triplicate samples (Student’s t-test). *P < 0.05, ***P < 0.001 and **** P < 0.0001

Fig. 5

TFRC overexpression partially rescues IGF2BP2 knockdown-mediated attenuation of colorectal cancer cell proliferation and iron metabolism. (A, B) After transfecting TFRC expression plasmids, the mRNA expression of TFRC and downstream factors and cell cycle-related factors were assessed using qRT-PCR. (C, D) Transfection of HCT-116 and SW480 cells expressing shIGF2BP2 with TFRC, and determination of protein levels using western blot. (E, F, G) Cell viability, clonogenicity, and proliferative capacity of shIGF2BP2-expressing HCT-116 and SW480 cells transfected or not transfected with oe-TFRC as assessed using the CCK-8 assay (E), colony formation assay (F), and EdU staining (G), respectively. (H) Assessment of plasmid-transfected HCT-116 and SW480 cells for TFRC via propidium iodide (PI) staining using flow cytometry. (I, J, K) Overexpression of TFRC in HCT-116 and SW480 cells transfected with shIGF2BP2 and determination of reactive oxygen species (ROS) concentration (I), total iron content (J), and labile iron pool (LIP) content (K). Each value represents the mean ± SD for triplicate samples (Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001

Fig. 6

IGF2BP2 enhances colorectal cancer tumor growth in vivo. (A, B) HCT-116 cells stably expressing shNC, shIGF2BP2 were subcutaneously injected into the right side of nude mice. Tumor images were obtained using the PerkinElmer IVIS preclinical in vivo imaging system at 25 (A) and 28 (B) days after injection. (C) Tumor volumes were measured after every 3–4 days starting on the 7th day after injection. (D) The weight of the xenografted tumors was measured. (E) Tumor sections were subjected to IHC staining with an antibody against Ki-67. Scale bar: 50 μm. (F) Tumor sections were subjected to IHC staining with an antibody against TFRC. Scale bar: 50 μm. (G) Kaplan-Meier survival curve shows the overall survival rate of mice in each group (n = 5, *p < 0.05, using the log-rank test). (H) A proposed regulatory model depicting the role of IGF2BP2 in iron metabolism and tumorigenesis (By Figdraw). Each value represents the mean ± SD for triplicate samples (Student’s t-test). **P < 0.01 and **** P < 0.0001